Title Page

Contents

CHAPTER I. INTRODUCTION 14

1. Metabolism of hyperthermophilic archaea 14

2. Carbohydrate utilization pathways in the hyperthermophilic archaea 19

3. Carbohydrate utilization pathways in Sulfolobus.species 25

4. Maltose/Maltodextrin metabolism of Sulfolobus species 28

5. The objectives of this study 30

6. References 32

CHAPTER II. DEVELOPMENT OF GENETIC SYSTEM IN SULFOLOBUS ACIDOCALDARIUS 41

1. Sulfolobus strains and culture conditions 41

2. Preparation of Sulfolobus medium 44

2.1. Liquid medium 44

2.2. Solid medium 45

3. Isolation of total DNA and RNA from S. acidocaldarius 47

4. Transformation of S. acidocaldarius 48

4.1. Methylationl of plasmid DNA 48

4.2. Preparation of Sulfolobus competent cells 49

4.3. Electroporation 52

4.4. Regeneration 52

4.5. Plating and incubation 53

5. Sulfolobus-E. coli shuttle plasmids 53

5.1. Replication origin 53

5.2. Selection system 56

5.2.1. Antibiotic selection 56

5.2.2. Uracil selection 70

5.3. Promoter for gene expression 85

6. Knockout systems 89

6.1. Gene disruption via PIL method 90

6.1.1. Construction of disruption vector pKHΔtrmBL1-K1 93

6.1.2. Construction of trmBL1 disruption mutant using pKHΔtrmBL1-K1 93

6.2. Gene disruption via DSC method 94

6.2.1. Construction of disruption vector pKHΔtrmBL1-K2 98

6.2.2. Construction of unmarked trmBL1 disruption mutant using pKHΔtrmBL1-K2 99

7. Summary 100

8. References 102

CHAPTER III. IDENTIFICATION OF THE MALTOSE/MALTODEXTRIN OPERON RELATED TO STARCH METABOLISM ON S. ACIDOCALDARIUS 109

1. Introduction 109

2. Materials and methods 120

2.1. Strains and culture conditions 120

2.2. DNA preparation and transformation 121

2.3. Sugar uptake measurements of S. acidocaldarius strains 121

2.4. Vector construction 122

2.5. Total RNA preparation and RT-PCR 126

2.6. Enzyme activity assay 127

2.7. Thin layer chromatography (TLC) 130

3. Results 130

3.1. Growth of S acidocaldarius DSM639 in the presence of starch 130

3.2. Identification of a putative gene cluster involved in starch metabolism of S. acidocaldarius DSM639 132

3.3. Transcription analyses of the genes in the putative maltose/maltodextrin operon in response to various sugars 136

3.4. Mutation of the ABC transporter genes in mal operon abolishes the growth of S. acidocaldarius on maltose, maltotriose, and starch 142

3.5. Identification of a TrmBL1-like protein involved in the transcriptional regulation of the genes in mal operon 148

3.6. Inactivation of trmBL1 in mal operon abolishes the growth of S. acidocaldarius in the presence of maltose and starch 152

3.7. TrmBL1 activates the expression of amylopullulanase and α-glucosidase genes during the growth on maltose and starch 155

4. Discussion 161

5. References 167

CHAPTER IV. CHARACTERIZATION OF AMYLOPULLULANASE IN THE MALTOSE/MALTODEXTRIN OPERON OF S. ACIDOCALDARIUS 173

1. Introduction 173

2. Materials and Methods 175

2.1. Strains and culture conditions 175

2.2. Construction of the apu overexpression vector 176

2.3. Construction of the apu disruption vector 177

2.4. Expression and purification of Apu in S. acidocaldarius 179

2.5. Enzyme assay 180

2.6. Hydrolytic patterns of Apu 182

2.7. Zymogram 183

3. Results 183

3.1. Sequence analysis of the putative amylopullulanase (Apu) 183

3.2. Identification of Apu critical for starch degradation in S. acidocaldarius 188

3.3. Homologous expression and purification of membrane-bound Apu 191

3.4. Catalytic properties of membrane-bound Apu 194

3.5. The role of Apu in starch metabolism of S. acidocaldarius 206

4. Discussion 209

5. References 215

CHAPTER V. CHARACTERIZATION OF MALA IN THE MALTOSE/MALTODEXTRIN OPERON OF S. ACIDOCALDARIUS 221

1. Introduction 221

2. Materials and Methods 224

2.1. Strains and culture conditions 224

2.2. Construction of the maLA overexpression vector 224

2.3. Expression and purification of MalA in S. acidocaldarius 227

2.4. Assay for α-glucosidase activity 230

2.5. Kinetic parameters of MalA 233

3. Results 233

3.1. malA induction and α-glucosidase activity of S. acidocaldarius grown in the presence of maltose and starch 233

3.2. Sequence analysis of MalA with other archaeal homologs 236

3.3. Expression and purification of MalA 239

3.4. Catalytic properties of MalA 240

3.5. Analysis of MalA activity by inactivation of MalEFG transporter 251

4. Discussion 253

5. References 261

CHAPTER VI. CONCLUSIONS 267

Abstract 277

Table 2.1. Sulfolobus strains used in this study and their characteristics 42

Table 2.2. Sulfolobus-E. coli shuttle plasmids constructed in this study 55

Table 2.3. Oligonucleotide primers used in this study 61

Table 2.4. Promoters used for target gene expression in Sulfolobus 86

Table 2.5. S. acidocaldarius MR31 derivatives constructed via PIL method 95

Table 3.1. Distribution of TrmB paralogues in the Thermococcales. 118

Table 3.2. Oligonucleotides used in this study 123

Table 3.3. Growth of S. acidocaldarius DSM639 on YT medium supplemented with various sugars 134

Table 4.1. Oligonucleotide primers used in this study 178

Table 4.2. Purification step of membrane-bound Apu produced from S. acidocaldarius MR31 harboring pKHapuA 196

Table 4.3. Effects of metal ions and reagents on the activity of Apu 200

Table 4.4. Substrate specificity of Apu towards various substrates 202

Table 4.5. Amino acid composition of thermophilic archaeal GH57 amylopullulanases 214

Table 5.1. Oligonucleotide primers used in this study 225

Table 5.2. Effects of metal ions and chemical reagents on the activity of MalA 247

Table 5.3. Substrate specificity of MalA 249

Table 5.4. Kinetic parameters of MalA towards various substrates 250

Table 5.5. Biochemical characteristics of thermophilic archeal α-glucosidases 258

Figure 1.1. Phylogenetic position of hyperthermophilic genera (thick lines) and a few moderate thermophilic and mesophilic archaeal genera (thin lines). 15

Figure 1.2. Pathways and reactions of organotrophic catabolism in the hyperthermophiles. 17

Figure 2.1. Solid culture of Sulfolobus using a gelrite plate. 46

Figure 2.2. The SuaI restriction and methylation system of S. acidocaldarius DSM639 (A) and the protection of plasmid against HaeIII digestion by the in vivo methylation (B). 51

Figure 2.3. Growth of S. acidocaldarius DSM639 in the presence of different concentrations of simvastatin. 59

Figure 2.4. Schematic diagrams for construction of PgdhA-hmg and Psac7d-hmg overexpression cassettes.(이미지참조) 68

Figure 2.5. (A) General mechanism of pyrimidine biosynthesis and genes related to the process in S. acidocaldarius. 73

Figure 2.6. Determination of MIC of 5-FOA, and UV exposure time for spontaneous mutation of S. acidocaldarius. 76

Figure 2.7. Mutation sites within pyrE gene in KH1U and KH2U mutants (A) and growth of S. acidocaldarius DSM639 and KH2U mutant on the 0.2% xylose medium in the presence or absence of uracil (10 ㎍/ml) (B). 80

Figure 2.8. Transformation of KH2U by introduction of Sulfolobus-E. coli shuttle vector pC. 83

Figure 2.9. Comparison of the strength of different promoters using β-glycosidase activity assay. 88

Figure 2.10. Schematic diagram to obtain trmBL1 disruption mutant using PIL method. 92

Figure 2.11. Schematic diagram to obtain trmBL1 disruption mutant using DSC method. 97

Figure 3.1. Schemes of starch (α-glucoside) metabolism in various hyperthermophilic archaea. 112

Figure 3.2. Operon organization of the genomic region around the maltose/maltodextrin ABC transporter genes in various hyperthermophilic archaea. 114

Figure 3.3. The growth curve (A) and starch degradation pattern (B) of DSM639 cells in YT medium containing 0.4% starch. 135

Figure 3.4. RT-PCR and β-glycosidase activity anaIysis of putative maltose/maltodextrin operon in S. acidocaldarius. 138

Figure 3.5. β-glycosidase activities of S. acidocaldarius MR31 harboring pKHlac2. 141

Figure 3.6. Growth of S. acidocaldarius DSM639 (●), MR31 (▲), and ΔmalEFG mutant (▼) in NYT and YT media supplemlented with different sugars. 144

Figure 3.7. Uptake of glucose (A), xylose (B), and maltose (C) by S. acidocaldarius DSM639 (●), MR31 (▲), and ΔmalEFG mutant (▼). 147

Figure 3.8. Multiple sequence alignment of S.acidocaldarius TrmBL1-like protein (Saci_1161). 150

Figure 3.9. Schematic diagram of domain structure and the amino acid sequence of maltose binding domain of Saci_1161. 151

Figure 3.10. Growth of S. acidocaldarius DSM639 (●), MR31 (▲), and ΔtrmBL1 mutant (■) in YT medium supplemented with different sugars. 154

Figure 3.11. Schematic representation of the mal operon (A) and sequence analysis of the promoter regions related to binding of TrmBL1 in mal operon (B). 157

Figure 3.12. α-Glucosidase (A) and α-amylase (B) activities on mal operon in S. acidocaldarius DSM639 (black bar) and ΔtrmBL1 mutant (gray bar). 159

Figure 4.1. Domain structure of Apu (A) and its comparison with other homologs of hyperthermophilic archaea (B). 186

Figure 4.2. Sequence alignment of Apu (Saci_1162) with S. tokodaii α-amylase (ST1102) and S. solfataricus α-amylase (Sso1172). 187

Figure 4.3. Growth of S. acidocaldarius DSM639 (●) and Δapu(▲) mutant in YT medium supplemented with starch. 190

Figure 4.4. TLC analyses of the hydrolyzed products of starch by membrane fraction of S. acidocaldarius DSM639 and Δapu mutant. 192

Figure 4.5. SDS-PAGE analysis (A) and zymogram (B) of purified Apu. 195

Figure 4.6. Effect of pH (A) and temperature (B) on the activity and stability (C) of Apu. 198

Figure 4.7. Hydrolysis patterns of Apu towards various substrates. 201

Figure 4.8. Hydrolytic activity of Apu towards starch. 205

Figure 4.9. Growth curve (●) and pH (口) change of S. acidocaldarius DSM639 during growth in YT medium supplemented with starch 208

Figure 5.1. Schematic diagram illustrating me PCR-based strategies used in construction of pKHmalA. 229

Figure 5.2. Determination of MalA activity in S. acidocaldarius DSM639 (gray bar) and ΔmalEFG mutant (black bar) grown in YT medium supplemented with various sugars. 235

Figure 5.3. The predicted primary structure of MalA expressed in S. acidocaldarius and its comparison with other related GH31 α-glucosidases. 238

Figure 5.4. SDS-PAGE analysis of purified MalA and molecular mass determination. 242

Figure 5.5. Effect of pH and temperature on the activity and stabiIity of MalA. 245

Figure 5.6. TLC analysis of hydrolysis patterns of MalA towards various substrates. 248

Figure 6.1. A model for function of the genes in mal operon of S. acidocaldarius DSM639. 276

 초고온성 미생물은 이들이 생산하는 효소들이 중온성 미생물 유래 효소들이 변성을 일으키는 온도에서 안정하게 효소반응을 수행하기 때문에 기존의 효소를 이용한 산업적인 단점을 극복할 수 있는 대안으로 관심이 증가되고 있다. 본 연구에서는 S. acidocaldarius를 연구모델로 삼아 genetic tool을 구축하고, 이를 바탕으로 전분 대사에 관여하는 단백질 및 효소들의 특성을 분석하고 전분 대사 관여 유전자들의 조절 기전을 분석하여 S. acidocaldarius의 전분 대사 기전을 규명하고자 하였다. 먼저 S. acidocaldarius의 안정적인 genetic tool을 개발하기 위해 이 고세균내에서 유전자를 안정적으로 발현하기 위한 발현 벡터를 구축하고, 형질전환방법을 최적화하였으며 이를 통해 유전체내 유전자가 파괴된 다양한 돌연변이주를 확보하였다. 구축된 genetic tool을 바탕으로 S. acidocaldarius의 유전체내 전분 대사에 관여하는 하나의 유전자군을 탐색하였으며 이 유전자군은 TrmB 조절 단백질과 유사한 단백질을 암호화하는 하나의 유전자(saci_1161)와 ABC transporter인 MalEFGK를 암호화하는 유전자들(saci_1163_64_65_66), 그리고 두 개의 아밀로오스 분해능을 갖는 효소를 암호화하는 유전자들(saci_1160과 saci_1162)을 포함하였다. 다양한 당의 첨가에 의한 이 유전자들의 발현을 RT-PCR와 lacS-fusion promoter assay를 통하여 이 유전자들의 RNA 발현은 말토오스와 전분의 첨가에 의하여 증가됨 확인하였다. 또한 ABC transporter가 결손된 돌연변이주(∆malEFG)의 다양한 당에서의 성장과 당 수송능의 변화 분석 결과 ∆malEFG 돌연변이주는 말토오스와 말토트리오스 그리고 전분에서 현저히 떨어지는 성장을 보였으며 말토오스에 대한 수송이 저해됨을 통해 탐색된 유전자군의 ABC transporter는 말토오스와 말토올리고당의 수송에 특이적인 단백질들임을 예측할 수 있었다.

이 유전자군 내에서 ABC transporter를 비롯한 다른 유전자들의 발현에 관여할 것이라 생각되어지는 하나의 조절 단백질(Saci_1161)이 탐색되었고 그 조절 단백질의 유전자군내에서의 영향을 알아보기 위해 조절 단백질이 결손된 돌연변이주(∆trmBL1)를 구축하였다. ∆trmBL1 돌연변이주의 다양한 당에서의 성장과 두 개의 아밀로오스 분해 효소들의 활성을 분석한 결과, 조절 단백질의 파괴는 말토오스와 전분에서 성장의 감소를 유발하고 말토오스와 전분이 첨가되는 조건에서 두 효소의 활성의 감소가 유도됨을 확인하였다. 이를 통해 탐색된 조절 단백질이 유전자군내 다른 유전자들의 발현에 영향을 미쳐 말토오스와 전분에서 성장을 억제시킨다고 예측 할 수 있었다. 최종적으로 유전자군내의 두 아밀로오스 가수분해 효소의 특성을 분석하였다. 유전자군 내 한 효소인 Saci_1162를 S. acidocaldarius 내에서 발현하였고 세포막으로부터 성공적으로 정제하였다. 정제된 단백질의 다양한 탄수화물에 대한 기질특이성 분석 결과 이 효소는 전분을 가수분해하여 말토오스, 말토트리오스와 포도당을 생성하는 amylopullulanse로 밝혀졌으며 100도에서 40시간 동안 활성을 유지하는 높은 열안정성을 가짐을 확인하였다. 그리고 유전자군 내 다른 amylolytic 효소인 Saci_1160의 기질특이성 분석 결과 이 효소는 α-glucosidase (MalA)로 판명되었고 세포내에서 dodecamer의 형태로 존재하며 말토오스와 짧은 사슬의 말토올리고당에 대한 높은 특이성을 가짐을 확인하였다. 이런 연구 결과를 통해, S. acidocaldarius는 전분을 amylopullulanase의 가수분해에 의해 말토오스와 말토올리고당으로 분해하고 이 분해된 당들이 ABC transporter를 통해 세포 내로 수송된 뒤, MalA의 작용에 의해 최종적으로 포도당으로 분해되어 에너지를 생성하도록 대사되는 것으로 보여진다.